Diagnostic array for aseptic encephalitis

描述（由申请人提供）： 摘要无菌性脑炎和脑膜炎是一种潜在的致命性疾病，其特征是大脑或覆盖大脑和脊髓的保护膜急性肿胀。这些疾病可以由病毒、细菌、真菌和寄生虫引起。病毒是脑炎最常见的原因，其中>85%的所有无菌性脑炎由肠道病毒引起。人类疱疹病毒占所有病毒感染的4%，西尼罗河病毒是美国最常见的虫媒病毒感染。另一方面，单纯疱疹病毒性脑炎如果不治疗，大约70%的病例会导致快速死亡。脑膜炎奈瑟氏菌可通过迅速扩散的点状皮疹与其他脑膜炎原因相鉴别，其他细菌和真菌脑膜炎/脑炎可通过脑脊液的生物化学和革兰氏染色容易地与病毒原因相鉴别。重要的是，CSF感染引起的并发症和适当的治疗策略取决于感染的类型和所涉及的物种。对于无菌性病因的疾病，病例适应性治疗因此依赖于快速诊断和区分病毒感染。十多年来，核酸扩增和检测试验一直被认为是CSF感染的首选试验，但只有三种FDA批准的病毒诊断试验，每种试验一次只能检测一种病毒。因此，本项目的目的是开发和试验一种用于诊断脑炎和脑膜炎的“封闭扩增子”凝胶元件RT-PCR阵列。该检测方法基于沃兹沃斯中心病毒病实验室开发的一套真实的实时PCR和逆转录酶PCR检测方法。第1阶段的性能目标是用于检测肠道病毒、疱疹病毒（HSV-1、HSV-2、VZV、CMV、HHV-6）和西尼罗河病毒的封闭扩增子RT-PCR阵列，分析LoD为d 50个基因组拷贝/反应。该检测试剂盒将包含外源性加标、定量提取和扩增对照品，该对照品在相同的多重反应中进行扩增和检测。在第1阶段中成功完成任何一个特定目标将保证第2阶段提案的重点是试剂稳定性、包装和有效期;临床前实验，以证明测定的重现性、重复性、灵敏度和特异性;以及开发用于测定数据分析和报告的自动算法和决策规则。 公共卫生相关性：封闭扩增子凝胶元件微阵列将是第一个将RT-PCR和微阵列技术结合在一个简单，廉价的检测试剂盒中用于诊断无菌性脑炎和脑膜炎。本文开发的基础平台同样将在传染病诊断的许多领域中得到广泛应用。

DESCRIPTION (provided by applicant): ABSTRACT Aseptic encephalitis and meningitis are potentially fatal diseases defined by acute swelling of the brain or protective membranes covering the brain and spinal cord. These diseases can be caused by viruses, bacteria, fungi and parasites. Viruses are the most common cause of encephalitis, with >85% of all aseptic encephalitis caused by enterovirus. Human herpes viruses account for 4% of all viral infections, and West Nile virus is the most common of the arboviral infections in the U.S. According to the CDC, most cases of human enteroviral encephalitis resolve without complication. One the other hand, herpes simplex encephalitis results in rapid death in approximately 70% of cases if left untreated. Neisseria meningitides can be differentiated from other causes of meningitis by a rapidly spreading petechial rash, and other bacterial and fungal meningitis/encephalitis can readily be differentiated from viral causes by biochemistry and gram staining of the cerebrospinal fluid. Importantly, complications arising from CSF infection and appropriate treatment strategies depend on the type of infection and the species involved. For aseptic causes of the disease, case-adapted therapy is therefore dependent upon rapidly diagnosing and differentiating between viral infections. Nucleic acid amplification and detection assays have been considered the test of choice for CSF infections for more than a decade, yet there are only three FDA-approved viral diagnostic tests, each of which only detects one virus at a time. The objective of this project is therefore to develop and trial a "closed amplicon" gel element RT-PCR array for the diagnosis of encephalitis and meningitis. The assay is based on a set of real- time PCR and reverse transcriptase PCR assays developed by the Laboratory of Viral Diseases at the Wadsworth Center. The performance goal for Phase 1 is a closed amplicon RT-PCR array for the detection of enterovirus, herpes viruses (HSV-1, HSV-2, VZV, CMV, HHV-6), and West Nile virus, with an analytical LoD of d 50 genome copies per reaction. The assay will contain an exogenously spiked, quantified extraction and amplification control which is amplified and detected in the same multiplex reaction. Successful completion of either Specific Aim in Phase 1 will warrant a Phase 2 proposal focused on reagent stability, packaging and shelf life; pre-clinical experiments to demonstrate assay reproducibility, repeatability, sensitivity and specificity; and developing automated algorithms and decision rules for assay data analysis and reporting. PUBLIC HEALTH RELEVANCE: The closed-amplicon gel element microarray will be the first-of-its-kind combination of RT-PCR and microarray technology in a simple, inexpensive test kit for the diagnosis of aseptic encephalities and meningits. The underlying platform developed herein will likewise find broad application in many areas of infectious disease diagnostics.